It is important to assay a quantity of a biological molecule, such as a protein or the like, not only for a fundamental research but also for a clinical diagnosis. For example, it is necessary and indispensable for an early diagnosis of a disease and an exact comprehension of a pathological condition to quantitate the quantity of the biological molecule, such as a cytokine, a hormone, a protein, a nucleic acid, or the like, that are included in body fluids, such as a plasma, a lymph, a tissue fluid, or the like, or to detect a specific biological molecule of very small quantity included in body fluids.
So far, an immunoanalytical technique (an immunoassay) is used for quantitating the above mentioned biological molecule, and it is able to provide as examples in principle a chemiluminescence method (a detection limit of 1.7×10−18 to 1×10−20 M), an enzyme-linked immunosorbent assay (ELISA) method (a detection limit of 1.7×10−15 to 1×10−18 M), a radioimunoassay (RIA) method (a detection limit of 1×10−15 M), a fluorescence method (a detection limit of 1×10−14 M), as technologies used for identifying an existence and a concentration of an analyte as an assay object in the immunoanalytical technique (the immunoassay) field. And, those are known as having a higher sensitivity in such the order (for example, refer to a nonpatent document 1).
Regarding the above mentioned ELISA method, an enzyme is used for a chromogenic reaction. Such the enzyme is not stable, and then there may be occurred a variation on a degree of chromophore due to a difference of enzyme activity between wells. Hence, sometimes it is not able to obtain data in high accuracy or in good reproductivity. Moreover, it takes time for a preparation of a substrate for an enzyme reaction or for a chromogenic reaction of the substrate by the enzyme reaction. And then long time period and complicated operations are required for obtaining an assay result. Hence, sometimes it is not able to process a large amount of specimen in a short time.
For resolving such the complication of the ELISA method, there is known the above mentioned fluorescence method using a reagent for labeling to be fluorescence labeled beforehand. Such the fluorescence method is convenient as the enzyme reaction is not included therein. However, the sensitivity is not sufficient, because a fluorescence intensity of a fluorescent molecule bound to one target substance is excessively small, because it occurs a self-quenching sometimes, and because a decoloration easily occurs regarding an organic dye to be used for labeling.
As the above mentioned immunoassay to detect or quantitate a biological molecule without using an enzyme reaction as well as the fluorescence method, an absorptiometric method is widely used for performing the detection and the quantitation of the biological molecule with a visual observation or by using such as an absorptiometer or the like, with binding a variety of light-absorbing substances to the biological molecule, such as a protein or the like, and with an absorption thereof as an indicator.
For such the light-absorbing substance, an organic molecule is mainly used. For example, an acrylamide gel after an electrophoresis is to be transcribed into a polyvinylidene difluoride (PVDF) film, it is stained using an Amido Black or a Coomassie Brilliant Blue (CBB) thereafter, and then an identification of a protein is to be performed by checking an absorption thereof with a visual observation. However, sometimes it is not able to perform the identification with a high reliability regarding a sample as a small quantity of not larger than one picomole by staining using the Amido Black or the CBB.
Regarding the immunoassay using the above mentioned absorptiometric method, a gold colloid immunoassay method is superior, that is for observing an absorption of a gold particle (for example, refer to patent document 1 and 2). However, the gold colloid is expensive and costly for such the gold colloid immunoassay method. Moreover, it is not able to use a variety of target particles using a difference of an absorption spectrum therebetween or a difference of an molar absorption coefficient therebetween, because an absorption wavelength is one type for a gold absorption, and because the molar absorption coefficient is constant as well. Hence, it is not able to apply it to an assay with a high throughput.
[Nonpatent Document 1] Kazutomo Imahori, Tamio Yamakawa, et al., “Seikagaku Jiten” the third edition (1998), P. 232, Tokyo Kagaku Dozin
[Patent Document 1] Japanese Patent Application Publication No. 2003-262638
[Patent Document 2] Japanese Patent Application Publication No. 1994-213891